DESCRIPTION: The exocrine pancreas represents an excellent system to study epithelial polarity, protein sorting, and regulated secretion. This application builds on the initial characterization of two zymogen granule membrane proteins, VAMP and GP2. VAMP (vesicle associated membrane protein) represents a family of integral membrane proteins that are essential to the vesicular transport of proteins. Studies supported by the initial First Award established that a protein similar to VAMP-2 found in synaptic vesicles is also present in the pancreatic zymogen granule. Further studies of VAMP, however, suggested a novel isoform is present in the pancreas. A cDNA clone for a new VAMP family member, currently named VAMP-3 has been isolated and the nucleotide sequence determined. A specific aim of this proposal is to characterize the subcellular the subcellular distribution of the VAMP-3 protein. Whether the subcellular distribution of VAMP-3 is distinct from the three other mammalian VAMPs previously characterized will be determined. Approaches to be used include subcellular fractionation, pulse chase studies, and microscopy. VAMP-3 exhibits a large intravesicular domain that differs from the other known mammalian VAMP isoforms. A potential function of this unique domain includes a role in VAMP sorting. In vitro mutagenesis will be used to generate chimeric proteins with the human transferrin receptor to determine whether the VAMP-3 unique domain can serve as a sorting signal. GP2 is the major membrane protein in the zymogen granule. Although its function is currently unclear, it is likely to be important in exocrine secretion. Recent data indicates that GP2 is processed intracellularly by endoproteolytic cleavage at the amino terminal end. Based on the hypotheses that intracellular processing regulates GP2 function, the sites for GP2 processing will be identified and mutated. These GP2 processing mutants will then be used in in vivo and in vitro studies to study GP2 function. Whether processing affects the ability of GP2 to bind to itself or to other zymogens will be determined. The role of protein processing in GP2 sorting will also be studied. Characterization of GP2 processing will lead to a better understanding of its function, sorting, and potential role in human diseases such as chronic pancreatitis.